1. Field of the Invention
The present invention relates generally to capillary columns having charged interior surface coatings and electrophoretic separation methods for their use. More particularly, the present invention involves methods for controlling electroosmotic flow in capillary electrophoresis columns having charged interior surface coatings by varying the concentration of buffer compounds which interact with the charged coatings.
2. Description of Relevant Art
Electrophoretic separation techniques have been utilized for years to separate molecules according to differences in the effective charge of the molecules, and/or according to differences in the molecular size of the molecules. Up until recently electrophoretic separations were conducted in gel slabs or open gel beds which were typically fabricated of polyacrylamide gel material. More recently capillary electrophoresis techniques combined with photometric detection methods have allowed the automation and rapid quantitative analysis of molecules. High revelation separations of molecules having different effective charges have been achieved by applying electrophoretic principles to buffer filled or gel filled narrow capillary tubes.
Typically, capillary columns used in capillary electrophoresis are fabricated of lengths of silica tubing having diameters on the order of 25 .mu.m to 200 .mu.m and lengths from about 10 to 200 cm. The buffer and gel separation mediums are pumped directly into the column interiors and electrophoretic techniques are used to separate numerous types of molecules including peptides, proteins, and oligonucleotides, nucleic acids and other charged molecular species. Moreover, the field is continually expanding with respect to the types and sizes of molecules which can be separated and detected using capillary electrophoresis procedures.
The advantages associated with capillary electrophoresis are numerous. Quantitative information can be achieved with very small sample sizes, and the amount of gel or buffer consumed is minuscule. Furthermore, the time required for the separations is sharply reduced, and the technique lends itself to automation and electronic data storage and data manipulation. Significantly, capillary electrophoresis is associated with certain phenomenon which are not present in tradition slab gel electrophoresis. One of these is the now familiar electroosmotic flow phenomenon characterized by bulk flow of buffer solutions toward one of the electrodes.
Electroosmotic flow is generated by the ionization of silanol functionalities on the surface of silica capillary tubing. The ionization results in a layer of protons in the electrophoretic buffer solution at the surface of the silica tubing. In the presence of an electric field the layer of protons resembles a positively charged column of fluid which migrates toward the cathode, causing a general bulk movement of the buffer medium. Advantageously, electroosmotic flow can be utilized in many applications to improve electrophoretic separations. For example, when the electrophoretic migration of the molecules being separated is in the opposite direction to that of electroosmotic flow, the net effect is an increase in effective column length.
Whether or not electroosmotic flow is advantageous for any particular separation, it is desirous to be able to control the flow. One method to minimize or to control electroosmotic flow, is to provide silica capillary tubing coated on the inside with a polymeric material in order to control the degree to which ionization of the surface silanol groups occurs. In general these coatings have served their intended purpose. However, there is an ongoing need for improved coatings and methods for using electroosmotic flow control to aid in electrophoretic separations.
Coated capillary columns are utilized typically to eliminate or substantially reduce electroosmotic flow during electrophoretic separation. However, interior surface capillary coatings can be designed so that separations using these coated capillaries actually have a characteristically higher electroosmotic flow compared with other coated or noncoated capillaries. In particular, for many applications, it would be desirous to utilize capillary electrophoresis columns having a particular coating and be able to increase or decrease the electroosmotic flow which is characteristic to that particular coated column.
The ability to vary electroosmotic flow in coated capillary column is especially advantageous when sample components are difficult to resolve and decreasing or increasing electrophoretic flow provides improved resolution. In cases where the sample components differ significantly in net charge, then increasing the electroosmotic flow can shorten the analysis time. If the sample components have similar net charge, then the ability to decrease the electroosmotic flow in order to enhance the small differences in the electrophoretic mobility is particularly advantageous.
Another problem associated with the capillary electrophoresis is the tendency for sample components to adhere to the wall of the capillary tubing, and in particular silica tubing. This is especially true in the case of small charged molecules which are easily attracted to reactive silica functionalities. When small peptides and amines are present in electrophoretic separation mediums, they interact. both electrostatically and hydrophobically with the capillary wall. The result is a decrease in separation efficiency and undesirable band broadening which gives erroneous separation data.
Coated capillary columns help minimize or eliminate undesirable capillary wall and sample interactions. For separating peptides and proteins, it has been suggested that charged polymeric coatings be applied. However, the ability to vary the electroosmotic flow using these coated capillary columns is minimal.
Accordingly, it is an objective of the present invention to provide methods for controlling electroosmotic flow in coated capillary columns.
It is additionally an objective of the present invention to provide methods for controlling electroosmotic flow in coated capillary electrophoresis columns and simultaneously reduce or eliminate interactions between sample components and the interior capillary wall.
It is additionally an objective of the present invention to provide capillary electrophoretic separation methods for the separation and resolution of a variety of charged molecules.